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ARTICLE OPEN Immunologic mechanisms of seasonal influenza administered by microneedle patch from a randomized phase I trial ✉ Nadine G. Rouphael1 , Lilin Lai2, Sonia Tandon1,3, Michele Paine McCullough1, Yunchuan Kong3, Sarah Kabbani1, Muktha S. Natrajan1, Yongxian Xu1, Yerun Zhu1, Dongli Wang1, Jesse O’Shea1, Amy Sherman1, Tianwei Yu3, Sebastien Henry4, Devin McAllister 4, Daniel Stadlbauer5, Surender Khurana6, Hana Golding6, Florian Krammer 5, Mark J. Mulligan7 and Mark R. Prausnitz 4,8

In a phase 1 randomized, single-center , inactivated influenza virus delivered through dissolvable microneedle patches (MNPs) was found to be safe and immunogenic. Here, we compare the humoral and cellular immunologic responses in a subset of participants receiving influenza vaccination by MNP to the intramuscular (IM) route of administration. We collected serum, plasma, and peripheral blood mononuclear cells in 22 participants up to 180 days post-vaccination. Hemagglutination inhibition (HAI) titers and antibody avidity were similar after MNP and IM vaccination, even though MNP vaccination used a lower dose. MNPs generated higher inhibition (NAI) titers for all three influenza virus vaccine strains tested and triggered a larger percentage of circulating T follicular helper cells (CD4 + CXCR5 + CXCR3 + ICOS + PD-1+) compared to the IM route. Our study indicates that inactivated influenza virus vaccination by MNP produces humoral and cellular immune response that are similar or greater than IM vaccination.

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INTRODUCTION Microneedle patches (MNPs) are micron-scale, solid, conical Each year, seasonal influenza epidemics cause 3–5 million severe structures made of dissolvable excipients or solid projections cases and 290,000 to 650,000 deaths globally1. More than half a coated with vaccine that deliver vaccine across the 14–18 million hospitalizations and 12,000 to 61,000 deaths occur in the stratum corneum into the viable epidermis and dermis . MNPs United States alone2. Seasonal influenza vaccination remains the provide an alternative to traditional IM injection of influenza virus 19 most effective way of preventing influenza . A better vaccine, especially for needle-phobic patients and offer multiple 20,21 understanding of the immunologic mechanisms of influenza virus potential advantages : simplicity (amenable to self-vaccination, vaccines3 is needed to overcome their limited effectiveness distribution, and storage outside the cold chain, such as on the (10–60%)4. Improving immune responses could be achieved by pharmacy shelf), potential for dose sparing, cost effectiveness higher antigen doses5, use of adjuvants6, or alternative routes of (reducing costs of vaccine administration, cold chain, and sharps immunization7. waste disposal)22, and safety (eliminating needle-stick injuries with Targeting the skin as a route of may be better at non-sharps waste). When tested in animal models23 and stimulating B and T cells than traditional vaccination routes by compared to other systemic influenza virus vaccine delivery utilizing the high density of dendritic cells and extensive modalities, the MNPs can induce higher neutralizing antibody microvascular and lymphatic networks facilitating migration of responses, higher protective immunity against lethal challenges of antigen-presenting cells to the regional lymph nodes8. Delivery to influenza viruses24, broader cross-reactive protection25, longer- the skin has been effective for many vaccines; most notably, the lasting protection25, and dose sparing effect26. use of scarification for resulted in the worldwide In 2015–2016, a first-in-human, phase 1 clinical trial on the use eradication of this highly virulent disease. In addition to the Bacille of a dissolvable MNP for trivalent seasonal influenza virus Calmette-Guérin vaccination against tuberculosis9, the intradermal immunization showed that the use of the MNPs was well route is also approved for the delivery of seasonal influenza virus tolerated, safe, had higher acceptability, and was strongly vaccine, which allowed for antigen sparing10,11 and improvement preferred by participants over conventional IM influenza vaccine of immunogenicity when equivalent doses of antigens have been administration27. The use of MNPs resulted in robust antibody compared (15 µg of HA per strain) between the intradermal and responses measured by hemagglutination inhibition (HAI) that intramuscular (IM) routes12. Novel skin delivery modalities have were at least as strong as IM injection for all vaccine strains, targeted the vaccine delivery both intradermally and transder- particularly for the influenza B virus strain. In that study, the mally13. However, to date, the use of skin delivery systems has immune response was characterized only in terms of HAI titers. been hampered by the lack of appropriate vaccine delivery Here, we report the exploratory results of a broader immunolo- systems combining reliability, safety, and simplicity of use. gical analysis of the phase 1 clinical trial in a subset of participants

1Hope Clinic of the Emory Vaccine Center, Division of Infectious Diseases, Department of Medicine, School of Medicine, Emory University, Atlanta, Georgia. 2Emory Vaccine Center, Department of Pediatrics, School of Medicine, Emory University, Atlanta, Georgia. 3Laney Graduate School, Emory University, Atlanta, Georgia. 4Micron Biomedical, Inc., Atlanta, Georgia. 5Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA. 6Division of Viral Products Center for Biologics Evaluation and Research, FDA, Silver Spring, MD, USA. 7New York University Langone Medical Center, Alexandria Center for Life Sciences, New York, NY, USA. 8School of Chemical & Biomolecular ✉ Engineering, Georgia Institute of Technology, Atlanta, Georgia. email: [email protected]

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Table 1. Demographic characteristics of participants (n = 22)a.

Characteristic MNP (n = 11) IM (n = 11)

Age, yearsb 27.5 (4.76) 28.5 (5.16) Sex, no. (%) Female 6 (55%) 5 (45%) Male 5 (45%) 6 (55%) Race, no. (%) White 6 (55%) 7 (64%) Black 4 (36%) 3 (27%) Other 1 (9%) 1 (9%) Ethnicity, no. (%) Non-Hispanic 11 (100%) 9 (82%) Hispanic 0 (0%) 2 (18%) BMI, kg/m2 25.1 (4.11) 25.6 (5.15) Prior IIV, no (%) 2013–2014 season 4 (36%) 1 (9%) 2012–2013 season 1 (9%) 3 (27%) Fig. 1 Fold change of hemagglutination inhibition (HAI) geo- metric mean titers (GMT) (log2) over baseline level at Day 28 and aNo statistical differences, assessed using Student’s t test for continuous Day 180 following vaccination against A/Christchurch/16/2010, variables and chi-square tests for categorical variables, were observed NIB-74 (H1N1), A/Texas/50/2012, NYMC X-223 (H3N2), B/Massa- between the groups for any variable. chusetts/2/2012, NYMC BX-51(B) strains for MNP (n = 11) and IM bContinuous variables are presented as mean (SD). (n = 11) groups with geometric standard error (vertical bars). BMI body mass index, IIV inactivated influenza vaccine. Black lines indicate statistical significance based on Wilcoxon rank- sum test. **P < 0.01. IM, IIV by intramuscular route; MNP, IIV by 1234567890():,; microneedle patch; D, Day. comparing the humoral and cellular immunologic mechanisms of inactivated influenza virus vaccine administered either using MNPs or hypodermic needles.

RESULTS Study participants, demographics From June 23rd to September 25th, 2015, 100 participants were enrolled and randomly assigned to study intervention. Detailed immunologic analyses were performed on the group receiving 2014–2015 inactivated influenza virus vaccine (IIV) by MNP (n = 11) versus IM route (n = 11), both delivered by a healthcare worker (Supplementary Fig. 1). The demographics of the groups were comparable: 50% of participants were female, 41% were non- white (Table 1). Ages ranged between 21 and 46, with a median of 26.5 years (IQR: 24.3, 32.0).

Serological immune responses We found that HAI responses to MNP and IM immunization were similar at 1 and 6 months after vaccination, and that HAI responses were higher at 1 month than at 6 months (Fig. 1, Fig. 2 Fold change of neuraminidase inhibition (NAI) geometric mean titers (GMT) (log2) over baseline level at Day 28 and Day Supplementary Table 1). There was no difference between the 180 following vaccination. Testing was performed against H6N1, MNP and IM groups in HAI geometric mean titers (GMTs) for the H6N2, and H6NB strains for MNP (n = 8) and IM (n = 11) groups with H1N1, H3N2, and B strains at baseline or 1 and 6 months after geometric standard error (vertical bars). Black lines indicate vaccination. The geometric mean fold rise (GMFR) was higher for statistical significance based on Wilcoxon rank-sum test. *P < 0.05; the B strain for the MNP group but not for other strains (P = 0.009). **P < 0.01. IM, IIV by intramuscular route; MNP, IIV by microneedle There was no difference in the Day 180-to-baseline GMT ratio patch; D, Day. between any strain or any group (H1N1: P = 0.33; H3N2: P = 0.29; B: P = 0.29). Seroprotection rates were similar between the groups H1N1 strain (27%), but the differences between MNP and IM with 100% at Day 28 for all strains and 82–100% when measured groups were not significant (P = 0.66). HAI responses in the 6 months after vaccination (P = 1.00). Seroconversion at Day 28 for substudy align with those previously observed in the complete 27 all strains varied between 55 and 82% for the MNP group and study dataset . between 18 and 82% for the IM group, but these rates were not While HAI has been routinely used to assess vaccine immuno- significantly different from each other (H1N1: P = 1.00; H3N2: P = genicity, lately neuraminidase inhibition (NAI) assays have been 1.00; B: P = 0.18). The seroconversion dropped remarkably at Day proposed as an independent correlate of protection28,29. Unlike 180 compared to Day 28 and was only present in some HAI measurements, NAI responses were often different after MNP participants in the MNP group for H1N1 (46%) and H3N2 (18%) vaccination compared to IM (Fig. 2, Supplementary Table 2). At strains, and only in a few participants in the IM group only for the baseline, there was no difference between the groups in NAI

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Fig. 3 Changes in antibody binding affinity at Day 28 post vaccination by vaccine delivery group (MNP: n = 10; IM: n = 11). SPR analysis of human plasma from various vaccine cohorts was performed with functional rHA1. Plasma antibody off-rate constants were determined as described previously in “Methods”. The box portion of the plot represents the interquartile range (IQR: 25th percentile, median, and 75th percentile) of the data. Error bars represent smallest and largest values within 1.5 times IQR. Black lines indicate statistical significance based on Wilcoxon rank-sum test. *P < 0.05; **P < 0.01. IM, IIV by intramuscular route; MNP, IIV by microneedle patch.

GMTs, with the exception of the N1 strain where the GMT for the IM group was higher than the GMT for the MNP group (P = 0.04) Fig. 4 Statistically significant fold changes in cytokines/chemo- (Supplementary Table 2). Interestingly, the fold changes between kines following vaccination. Fold changes at a Day 2 and b Day 8 Day 28 and baseline for NAI GMTs were higher for N1 (P = 0.002), post vaccination by vaccine delivery group (MNP: n = 8; IM: n = 11). N2 (P = 0.003), and B (P < 0.001) strains after vaccination by MNP The proteins were detected by antibody-bound beads and quantitated using a Luminex instrument. The box portion of the when compared to IM (Fig. 2, Supplementary Table 2). These = = plot represents the interquartile range (IQR: 25th percentile, median, results were also observed at Day 180 (N1: P 0.05; B: P 0.02) and 75th percentile) of the data. Error bars represent smallest and with the exception of the N2 strain (P = 0.11). largest values within 1.5 times IQR. Black lines indicate statistical We also measured antibody affinity by analyzing the significance based on Wilcoxon rank-sum test. *P < 0.05; **P < 0.01. antigen–antibody complex dissociation kinetics (off-rates) bound IM, IIV by intramuscular route; MNP, IIV by microneedle patch. to H1N1pdm09 HA1 (globular head). Antibody affinity could be of clinical significance as high levels of low avidity antibodies in infected individuals were associated with severe H1N1pdm09 DR+ cell subset. No significant difference was observed between disease30. Pre-vaccination off-rates varied among participants = −2 −1 −3 −1 the groups (P 0.33) (Fig. 5). ranging between 10 s and 10 s (Fig. 3) and were We looked at polyfunctional CD4+ T cells producing different = comparable between groups (P 0.65). Increase in binding cytokines such as IL-2, IL-21, IFN-γ, and CD154 (CD40L). Relative to fi af nities was observed within both the MNP and IM groups at baseline, there was a numerical increase in the median percentage = Day 28 post vaccination when compared to baseline (P 0.002; of vaccine-induced IL-2 + CD40L + CD4+ T cells at Day 8 in the = P 0.01, respectively). MNP group [0.32% (IQR: 0.16, 0.37)] compared to the IM group [0.11% (IQR: 0.06, 0.21)], but it was not statistically significant (P = Cytokines/chemokines 0.07) (Fig. 6). No significant change of IFN-gamma, TNF-alpha, and An increase was seen in IP-10 (P = 0.01) for the MNP group IL-21 was detected over baseline. compared to the IM group when measured on Days 2–3 after Prior studies have shown a transient increase in cTFH cells immunization and compared to baseline. However, for TNF-α (P = measured 7 days after influenza virus vaccination correlating with 0.04), the level was higher for the IM group compared to the MNP the emergence of antibody-secreting cells, increased levels of serum group at Days 2–3 when compared to baseline. When Day 8 antibody titers and affinity, as well as memory B cell responses31–33. values were compared to baseline for the following cytokines: IL-8 These T cells express the chemokine receptor CXCR5 and co- (P = 0.02); IL-5 (P = 0.034); IL-13 (P = 0.013) and MIP-1b (P = 0.016) stimulatory molecules ICOS and PD-1, and thus belong to a they were all higher for the MNP group (Fig. 4). After adjusting for circulating compartment of TFH cells. There was a higher percentage multiple comparisons via Bonferroni correction, none of these of cTFH (CD4 + CXCR5 + CXCR3 + ICOS + PD-1+) at Day 8 post findings remain statistically significant. Other changes in cytokines vaccination in the MNP group [2.48% (IQR: 1.9, 6.8))] compared to and chemokines over baseline values did not differ significantly the IM group [0.96% (IQR: 0.8, 2.5)] (P = 0.04) (Fig. 7). between groups, including IL-6 and MIP-1 alpha. Influenza virus-specific immunoglobulin G (IgG)-secreting memory B cells (MBCs) responses were measured at baseline Cellular immune responses and at Day 28 without any significant difference between the Proinflammatory monocytes in blood were defined as CD14 + groups for all 3 antigens: H1 (P = 0.91), H3 (P = 0.55), and N2 (P = CD16+ monocytes at Days 2–3 within the CD3-CD19-CD56-HLA- 0.15) (Fig. 8).

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Fig. 5 CD14 + CD16+ monocytes after vaccination. a Monocytes were identified within the SSC-A-hi FSC-A-hi cells as the CD3-CD19-CD20- CD56- HLA-DR+ population and gated for the CD14 + CD16-, CD14 + CD16+, and CD14dimCD16++ subsets. b Temporal CD14 + CD16+ monocytes frequency over CD3-CD19-CD56-HLA-DR+ cells at baseline, 2–3, 8, and 28 days post vaccination. c Percentage of CD14 + CD16+ monocytes at Days 2–3 on CD3-CD19-CD56-HLA-DR+ cells by vaccine delivery group (MNP: n = 7; IM: n = 10). The box portion of the plot represents the interquartile range (IQR: 25th percentile, median, and 75th percentile) of the data. Error bars represent smallest and largest values within 1.5 times IQR. IM, IIV by intramuscular route; MNP, IIV by microneedle patch.

DISCUSSION CD4+ T cells response at Day 8 relative to baseline when MNPs have the potential to offer many benefits to immunization compared with the response seen with IIV administered IM. programs including decreased transport and distribution costs In addition, the interactions between CD4+ T cells and B cells (thermostable, small footprint), ease and safety of administration promote both extrafollicular responses that generate short-lived, (no need for reconstitution, no specialized skills), and greater class-switched antibody-secreting cells as well as germinal center 17 fi acceptability and potentially less hesitancy by end-users . While reactions through cTFH that result in long-lived, af nity-matured 46 immunologic advantages of MNPs as a novel vaccine delivery antibody-secreting cells and memory B cells . method have been demonstrated in animal models, the current There are limitations to this study. A major limitation is the small study is one of few to highlight the unique immunologic features group sizes typical for a phase 1 study and availability of samples. of MNPs in humans in the setting of influenza virus vaccination, The lack of difference in HAI GMT between the different delivery other than dose sparing18,34. routes could have been due to pre-existing antibodies linked to Immune responses induced by available IIV vaccines primarily reduced humoral and effector B cell responses after vaccina- 47,48 target the immunodominant globular head domain of the HA tion making it difficult to compare our results with other which is responsible for viral attachment and fusion. The HAI studies where the majority of the participants had lower pre- assay, measuring the antibodies to the HA head, is considered a existing antibodies18,34. Detecting differences in immune profiles surrogate correlate of protection and used in the evaluation of between the different routes of delivery might have been easier in vaccines for licensure35; however, an HAI titer of 40 only provides naive individuals (young children)49, where strong immunologic a 50% putative protective threshold in adults36. In addition, HA advantages of influenza vaccination by MNP compared to the IM head-specific antibodies do not reflect the whole spectrum of route have been seen in a young mouse model50. protective antibodies. Blocking neuraminidase (NA), another major There was a difference between the HA quantity in the vaccines surface glycoprotein, is an effective way to inhibit viral infection delivered by IM versus MNPs, with 15–37% lower quantities replication and shedding37. Antibodies against NA have been delivered by MNPs27. Despite this, MNP delivery still showed shown to confer protection from natural influenza virus infec- similar HAI as well as superior NAI and cTFH cell responses tion28,29 as well as in an influenza challenge model38 and to highlights another advantage of MNPs with regard to antigen shorten the duration of shedding and symptoms39. Therefore, NAI sparing. While the HA quantity in seasonal influenza virus vaccine assay is being considered as an independent correlate of is at least 15 µg per strain, the quantity and quality of NA included protection40,41. Interestingly, the MNPs were able to induce a in the vaccine are not standardized51. We measured the quantity better NAI responses at Days 28 and 180 when compared to of HA administered by the MNPs, but did not measure the baseline in comparison to the IM route. quantity of NA in either vaccine type, though both the IM doses Intradermal vaccination directly targets epidermal Langerhans and the monobulk used to prepare the MNPs were provided by cells and dermal dendritic cells, which are essential for efficient the manufacturer, so we would expect the ratio of HA-to-NA to be cellular and humoral responses42. In our study, MNPs showed an the same in MNP and IM formulations. early increase in IP-1043, MIP-1b, and IL-8 compared to baseline Memory B cell responses in our study were measured early, that was statistically higher than the IM route with a trend toward 28 days after vaccination, and did not show any difference a higher proinflammatory monocyte responses 2–3 days after between the groups; assessing these responses at a later vaccination. Interestingly, higher levels of allergic inflammation IL- timepoint (4–6 months) may have revealed differences in the 5 and IL-13 were noted in the MNP group, which could explain the groups. In addition, CD8+ T cells play an important role in local transient pruritus reported by a subset of vaccinees27. influenza virus immunity. Due to limitation in availability of Though antibodies have been the hallmark of protection peripheral blood mononuclear cells (PBMCs) in our samples, we against influenza vaccines and infection, CD4+ T cells confer were unable to conduct these assays. protection too44,45. Pre-existing CD4+ T cells result in lower virus In addition, an extended immunological analysis was not shedding and less severe influenza symptoms in experimental A/ performed on any subject who received the placebo MNP in the H3N2 human challenge44 and natural infection with A/ trial. It is not possible therefore to determine if any of the H1N1pdm45 in the absence of protective antibodies. In our study, responses observed were due to the application of the dissolving MNPs did not lead to a better HA-specific IL-2 + CD154(CD40L) + MNP rather than being due to delivery of the influenza antigen.

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Fig. 6 IL-2 secreting CD4+ T cells after vaccination. a Representative FACS plot showing CD3 + CD4+ T cells expressing CD154 (CD40L) and IL-2 by ICS assay. b Percentage of vaccine-induced IL-2 + CD40L + CD4+ T cells at Day 8 by vaccine delivery group (MNP: n = 8; IM: n = 8). The box portion of the plot represents the interquartile range (IQR: 25th percentile, median, and 75th percentile) of the data. Error bars represent smallest and largest values within 1.5 times IQR. IM, IIV by intramuscular route; MNP, IIV by microneedle patch.

In summary, the use of MNPs for influenza virus vaccination MNPs used in the substudy showed that the mean HA dose delivered by offers both logistical advantages as well as promising immuno- MNPs was 10.8 μg (Standard Error (SE) 1.1) for the H1N1 strain, 14.4 μg (SE logic advantages reported here and elsewhere. Our study showed 0.8) for the H3N2 strain, and 13.3 μg (SE 0.6) for the B strain27. Our higher cT cells along with higher NA function in participants measurements also showed that the IM injection administered 18 μgofHA FH μ μ vaccinated with MNPs when compared to the traditional IM per H1N1 vaccine strain, 17 g of HA per H3N2 vaccine strain, and 15 gof HA per B vaccine strain with a delivery efficiency of 60%, 85%, and 89%, needle and syringe. respectively. Mean dose delivered by each strain significantly differed between MNP and IM groups (H1N1: p < 0.001; H3N2: p = 0.013; B: p = 0.018). METHODS Participants Blood samples The partially blinded, randomized, placebo-controlled, phase 1 study Blood samples for the substudy were collected at Days 0 (pre-vaccination), recruited 100 healthy non-pregnant, immunocompetent adults aged – – – – 18–49 years who had not previously received the influenza virus vaccine 2 3, 8 10, 26 30, and 166 194 days post vaccination. Peripheral blood during the 2014–15 influenza season. Additional inclusion and exclusion mononuclear cells (PBMCs) were separated from sodium citrate whole criteria are detailed on ClinicalTrials.gov (NCT02438423; date of registra- blood using a Ficoll-Hypaque gradient and stored in a liquid nitrogen tank. tion: May 8, 2015) and have been previously described elsewhere27. Cryopreserved PBMCs were thawed in a 37 °C water bath and washed. Before use, cells were counted and checked for viability by Trypan blue dye exclusion. Randomization and study groups Participants, who consented for the substudy, were randomly assigned to Hemagglutination inhibition assay receive either IIV by MNP or by IM injection, in each case administered by a – fl healthcare worker. In addition, there was a group that received placebo by We used reference strains contained in the 2014 15 trivalent in uenza MNP and another group that received IIV by MNP self-administered by virus vaccine: H1N1 virus reference strain A/Christchurch/16/2010 (#10/ study participants. As the main objective of the phase 1 trial was to 216, National Institute for Biological Standards and Control, Potters Bar, compare IIV vaccination by MNP and IM, both applied by healthcare Hertfordshire, UK), H3N2 A/Texas/50/2012 (#FR1210), and B virus reference fl worker, the substudy compared these two groups for detailed immuno- strain B/Massachusetts/2/2012 (#FR1196, In uenza Reagent Resource of logic analysis (Supplementary Fig. 1). Laboratory testing was performed in the Centers for Disease Control and Prevention, Atlanta, GA, https://www. fl a blinded manner. internationalreagentresource.org). In uenza viruses were propagated in Madin-Darby Canine Kidney (MDCK).2 cells and MDCK.2 SIAT1 cells in the presence of tosylamide-2-phenylethyl chloromethyl ketone-treated trypsin Vaccines (Sigma-Aldrich, St Louis, MO). HAI assays were previously performed and The licensed 2014–15 seasonal trivalent IIV (Fluvirin) was kindly provided described27. by Seqirus (Cambridge, MA) in single-dose, prefilled syringes for IM fl injection containing the following three in uenza vaccine strains: A/ Neuraminidase inhibition assay Christchurch/16/2010, NIB-74 (H1N1); A/Texas/50/2012, NYMC X-223 H6NX reassortant influenza A viruses were generated by reverse genetics (H3N2); and B/Massachusetts/2/2012, NYMC BX-51(B). The MNPs were 53 designed at the Georgia Institute of Technology (Atlanta, GA) and as described previously . These viruses contain the HA (H6) gene from fl manufactured by the Global Center for Medical Innovation (Atlanta, GA) in uenza A/turkey/Massachusetts/3740/1965 (H6N2) virus, gene segments fl under Phase 1 Good Manufacturing Practice. The antigen incorporated into encoding internal proteins from in uenza A/Puerto Rico/8/1934 (H1N1) the MNPs was also provided by Seqirus and comprised the same three virus, and one of the following NA gene segments: N1 of A/California/04/ 2009 (H1N1); N2 of A/Texas/50/2012 (H3N2) or influenza B NA of B/ vaccine strains. The formulation and fabrication methods have been 54 previously described52. Yamagata/16/1988 . H6NX viruses were propagated in 10-day-old fi IM was administered by hypodermic needle in the deltoid muscle of the speci c-pathogen-free embryonated eggs at 37 °C for 2 days. arm preferred by the participant and the MNPs were manually applied to To determine the ideal stock virus concentration to be used in the NAI fi the dorsal aspect of the wrist of the non-dominant arm and left on the skin assay, an enzyme-linked lectin assay (ELLA) was rst performed for all for 20 min. reassortant H6NX viruses. In brief, 96-well microtiter plates (Thermo Fisher, Waltham, MA) were coated with 100 μL/well fetuin (Sigma) at a concentration of 25 μg/mL in PBS and stored at 4 °C. The next day, the Assessment of vaccine delivery plates were washed three times with T-PBS [PBS containing 0.1% Tween-20 The difference between the antigen content measured by single radial (Sigma)] and blocked in 200 μL blocking buffer (5% bovine serum albumin immunodiffusion (SRID) assay of unused MNPs (i.e., 18 μg (BSA) (MP Biomedicals, Irvine, CA) in PBS) at room temperature for at least (HA) per vaccine strain) and that of the residual antigen content of the 11 1 h. On a separate 96-well plate, virus was serially diluted 1:2 in

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Fig. 7 Circulating T follicular helper (cTFH) cells after vaccination. a Representative FACS plot showing ICOS and PD-1 co-expressing CD3 + CD4 + CXCR5 + CXCR3 + cTFH cells. b Temporal cells at baseline and days 2–3, 8, and 28 post vaccination. c Percentage of cTFH (CD4 + CXCR5 + CXCR3 + ICOS + PD-1+) at Day 8 post vaccination by vaccine delivery group (MNP: n = 8; IM: n = 9). The box portion of the plot represents the interquartile range (IQR: 25th percentile, median, and 75th percentile) of the data. Error bars represent smallest and largest values within 1.5 times IQR. Black lines indicate statistical significance based on Wilcoxon rank-sum test. *P < 0.05; **P < 0.01. IM, IIV by intramuscular route; MNP, IIV by microneedle patch.

PBS and added to the fetuin-coated plate. The plates were incubated for domains could also adsorb the majority of neutralizing antibodies from 2 h at 37 °C and washed four times with T-PBS. Peanut agglutinin post-vaccination polyclonal antibodies in human plasma. conjugated to horseradish peroxidase (PNA-HRP, Sigma) at a concentration Steady-state equilibrium binding of post-vaccination sera was mon- of 5 µg/mL diluted in PBS was added to the plate (100 µL per well) and itored at 25 °C using a ProteOn surface plasmon resonance biosensor (SPR, incubated in the dark for 1.5 h at room temperature. The plate was Bio-Rad, Hercules, CA). The rHA1 protein is coupled to a GLC sensor chip developed with 100 µL o-phenylenediamine dihydrochloride (SigmaFast) with amine coupling with 500 resonance units (RU) in the test flow cells. and the reaction stopped after 5 min with 50 μL 3 M hydrochloric acid Samples prepared at 10-, 50-, and/or 250-fold dilutions were injected at a (Thermo Fisher). The plate was read at a wavelength of 490 nm with a flow rate of 50 µL/min (120-s contact time) for association, and dissociation microtiter plate reader (Bio-Tek, Winooski, VT). The data were fitted to a performed over a 600 s interval (at a flow rate of 50 µL/min). Responses nonlinear regression curve (four parameters) to determine the effective from the protein surface were corrected for the response from a mock concentration 50 (EC50) of the different viruses. surface and for responses from a separate, buffer-only injection. MAb 2D7 Working stocks of each H6NX virus were prepared by diluting the virus (anti-CCR5) was used as a negative control. Antibody off-rate constants, stock to two times the EC50. which describe the fraction of antigen–antibody complexes that decay To perform the NAI assays, the microtiter plates were coated and per second, were determined directly from the plasma sample interaction blocked as described above. In the first row of a cell culture 96-well plate, with rHA1 using SPR in the dissociation phase only for the sensorgrams 150 µl of 1:20 pre-diluted and pre-treated human serum was added. The with Max RU in the range of 20–150 RU and calculated using the BioRad human sera were pre-treated with RDE (Denka Seiken, Tokyo, ) to ProteOn manager software for the heterogeneous sample model as reduce non-specific background inhibition. In all other wells, 75 µl PBS was described before. Off-rate constants were determined from two indepen- added. The sera were serially diluted two-fold by transferring 75 µl into the dent SPR runs. Sample sizes available for statistical analysis differed based next row. Then, 75 μL of virus working stock (2× EC50) was added to wells on sample availability (MNP: n = 10; IM: n = 11). of the serially diluted human sera and incubated for 1.5 h on a shaker. The fetuin-coated plates were washed three times with T-PBS and the virus/ serum mixture was added to the plates and incubated for 2 h at 37 °C. The Innate and T-cell phenotyping assays remainder of the assay was performed as described above. The data were Monoclonal antibodies used for staining of PBMCs for innate cell fitted to a nonlinear regression curve (four parameters) and the inhibitory phenotyping were CD3 (UCHT1, #557943), CD19 (HIB19, #557921), CD14 concentration 50 (IC50) was calculated for all human serum samples (M5E2, #565283), HLA-DR (G46-6, #560651), CD11c (O33-782, #561355), individually. Sample sizes available for statistical analysis differed based on and CD123 (7G3,554529) from BD Biosciences (Franklin Lakes, NJ), CD56 sample availability (MNP: n = 8; IM: n = 11). (MEM188,17-0569) and CD16 (CB16,47-0168) from eBiosciences (San Diego, CA); for cTFH phenotyping CD3 (SP34-2, #562877), CD4 (L200, #560836), CXCR5 (RF8B2), and CXCR3 (IC6/CXCR3) BD Biosciences, ICOS Serum antibodies binding assay (C398.4 A) and PD-1 (EH12.2H7) from Biolegend (San Diego, CA) The DNA gene segments corresponding to the HA1 proteins of A/ (Supplementary Fig. 2). Data were collected on an LSRII (BD Biosciences) California/07/2009 (H1N1pdm09) virus were cloned as NotI-PacI inserts into and data were analyzed using R 3.6.3 software. Sample sizes available for a T7 promoter based pSK expression vector in which the desired statistical analysis differed based on sample availability (monocytes—MNP: polypeptide can be expressed as a fusion protein with His6 tag at the C- n = 7; IM = 10, cTFH—MNP: n = 8; IM: n = 9). terminus. E. coli Rosetta Gami cells (Novagen, Madison, WI) were used for expression of HA1 proteins. Following expression, inclusion bodies were fl fi + isolated by cell lysis and multiple washing steps, denatured, refolding in In uenza virus-speci c CD4 T cells by intracellular cytokine redox-folding buffer, and dialyzed. The dialysate was filtered through a assay (ICS) 0.45 µm filter and purified by HisTrap Fast flow chromatography Peptide pools consisting of 15-mers with 11 amino acid overlaps spanning (information on instrument/materials). The purified proteins were char- 3 HA proteins in the vaccine were synthesized (GenScript, Piscataway, NJ). acterized by the presence of oligomers by gel-filtration chromatography Cryopreserved PBMCs were thawed in a 37 °C water bath and washed. and by functional binding to turkey RBCs in a hemagglutination assay. Cells were counted and checked for viability by Trypan blue dye exclusion To confirm that the rHA1 proteins formed oligomers (similar to the and rested overnight at incubator 37 °C with 5% CO2 and then incubated native spike HAs on virions) hemagglutination assay was performed with with virus peptide pools at final concentrations of 2 μg/mL of each peptide human RBCs. The HA0 from H1N1pdm09 and the purified rHA1 proteins in the presence of CD28 and CD49d (BD Biosciences, #340957 and agglutinated RBC to various concentrations. We previously demonstrated #340976). Negative control samples were left un-stimulated in culture that several recombinant HA1 domains produced using bacterial system medium, and positive control samples were treated with Staphylococcus resembled native viral HA in EM, formed functional trimers/oligomers that enterotoxin B (Sigma) at a final concentration of 1 μg/mL. Cells were were fully immunogenic, generated high-affinity antibodies, and protected cultured for 2 h at 37 °C, and then a cocktail containing brefeldin A and ferrets from influenza challenge with strains55–58. The HA1 monensin was added (eBioscience, #004980-93), followed by a 4 h culture.

npj Vaccines (2021) 89 Published in partnership with the Sealy Institute for Vaccine Sciences N.G. Rouphael et al. 7

Fig. 8 Influenza-specific IgG-secreting memory B cells (MBCs) after vaccination. a Representative ELISpot showing H1 protein and IgG protein-specific memory B cells at baseline and Day 28 post vaccination. b Fold change of influenza-specific IgG-secreting Memory B Cells (MBCs) responses at Day 0 and Day 28 by vaccine delivery group (MNP: n = 10; IM: n = 11). The box portion of the plot represents the interquartile range (IQR: 25th percentile, median, and 75th percentile) of the data. Error bars represent smallest and largest values within 1.5 times IQR. IM, IIV by intramuscular route; MNP, IIV by microneedle patch.

Cells were washed with 1xPBS; surface stained with Aqua live/dead stain Cytokines and chemokines L423102 (Biolegend), and then fixed and permeabilized using a Cytofix/ Cytokines tested included IL-1β, IL-1RA, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-7, IL-8, Cytoperm kit (BD Biosciences; 554722). The cells were then stained with IL-10, IL-12p40/p70, IL-13, IL-15, IL-17, TNF-α, IFN-α, IFN-γ, GM-CSF, MIP-1α, the following fluorescence conjugated antibodies: CD3(SP34-2), CD4(L200), MIP-1β, IP-10, MIG, Eotaxin, RANTES, and MCP-1. Human 25-plex cytokine IL-2 (MQ1-17H12, #554567), and TNF-α (MAB11, #550679) from BD kits (Cat # LHC0009M) were purchased from Thermo Fisher (Waltham, MA) Biosciences; and IFN-γ (4 S.B3, #47-731942) and IL-21 (2A3-N2) from for Luminex assays and used according to the manufacturer’s recommen- eBioSciences. After the cells were washed, data were collected on an LSRII dations with modifications. Beads were added to a 96-well plate and Fortessa instrument (BD Biosciences). Compensation was performed using washed in a Biotek ELx405 washer. Samples were added to the plate tubes of CompBeads (BD Biosciences, # 51-90-9001229) individually containing the mixed antibody-linked beads and incubated at room stained with each fluorophore and compensation matrices were calculated temperature for 1 h followed by overnight incubation at 4 °C. Cold and with FACSdiva. Data were analyzed using Flowjo software. Sample sizes room temperature incubation steps were performed on an orbital shaker available for statistical analysis differed based on sample availability (MNP: at 500–600 rpm. Following the overnight incubation, plates were washed n = 8; IM: n = 8). in a Biotek ELx405 washer and then biotinylated detection antibody added for 75 min at room temperature with shaking. Plates were washed again as Memory B cell analysis by enzyme-linked immune absorbent above and streptavidin-PE was added. After incubation for 30 min at room spot (ELISpot) assay temperature, wash was performed as above and reading buffer was added to the wells. Each sample was measured in duplicate. Plates were read fl Recombinant in uenza virus HA proteins of A/Christchurch/16/2011 using a Luminex LX 100 instrument with a lower bound of 50 beads per (H1N1) pdm09 (NR-42487) and neuraminidase (NA) Protein from A/ sample per cytokine. As cytokine changes are observed early after Brisbane/10/2007 (H3N2) (NR-43784) were provided by BEI Resources vaccination, we measured cytokine levels 2–3 days and 8–10 days after (Manassas, VA). HA of A/Victoria/361/2011/H3N2 (FR-1059) was provided vaccination and compared them to pre-vaccination levels. Sample sizes by the Centers for Disease Control and Prevention International Reagent available for statistical analysis differed based on sample availability (MNP: Resource (Manassas, VA). Cryopreserved PBMCs were thawed and checked n = 8, IM: n = 11). for viability as described above. Memory B cell assays were performed as previously described59. In brief, PBMCs were plated in 24-well dishes at 5 × 105 cells/well in R-10 medium supplemented with IL-2 and R488 (CTL- Statistics hBPOLYS-200, CTL) plus antigens for 6 days. For ELISpot, 96-well filter Descriptive statistics of the population at enrollment were compared using plates (Millipore, #MSHAN4B50) were coated 0.1 µg per well of HA proteins Student’s t test for continuous variables and chi-square tests for categorical or 1 µg per well of anti-human IgG. The plates were left to adsorb variables. The immunogenicity population included all participants who overnight at 4 °C, washed four times in PBS, and incubated in Roswell Park provided serum samples at baseline and at least 28 or 180 days after study Memorial Institute (RPMI) medium with 10% (vol/vol) fetal bovine serum product administration (MNP: n = 11, IM: n = 11). GMT of HAI and NAI (FBS) for 1 h at 37 °C. RPMI was removed, and PBMCs suspended in RPMI antibodies were determined for each strain and 95% confidence intervals with 10% FBS were placed in each well with threefold dilutions. Plates were (CI) were based on the normal distribution of log-transformed data. incubated at 37 °C overnight, then were washed twice with PBS followed Mann–Whitney Wilcoxon tests were used to compare MNP and IM groups. by four times in PBS with Tween® 20 (PBST) and incubated for 2 h at room We adjusted for multiple comparisons using Bonferroni correction. temperature with biotinylated anti-human IgG (Jackson Immunoresearch The proportion of participants achieving seroprotection (defined as a Laboratory, #709065098) diluted 1:1000 in PBST with 2% FBS. Plates were HAI antibody titer of 1:40 or greater) and seroconversion (defined as a washed four times in PBST and incubated for 1 h at room temperature with minimum four-fold increase in post-vaccination HAI and NAI antibody titer) streptavidin-HRP (Vector Laboratories #A 2004) diluted 1:1000 in PBST with ~28 days following receipt of study products were determined for each 2% FBS. Plates were washed four times each in PBST, then with PBS, strain in the MNP and IM groups. Clopper-Pearson exact confidence followed with incubation with 3-Amino-9-ethylcarbazole substrate kit intervals were calculated, and chi-square tests were used to compare (EMD Millipore Corporation, Substrate #152226, Buffer #152224) for 10 min frequencies of seroprotection and seroconversion between each group. until spot development. Plates were washed with water and allowed to For all cellular assays, median percentage and IQR were reported and dry; images were obtained using a CTL ELISpot plate reader. Data are Mann–Whitney Wilcoxon tests were used to compare MNP and IM groups. presented as the percentage of influenza virus-specific IgG-secreting cells All statistical analyses were performed using the R statistical software among total IgG-secreting cells. Sample sizes available for statistical version 3.6.3 and a two-sided p-value of <0.05 was considered statistically analysis differed based on sample availability (MNP: n = 10, IM: n = 11). significant.

Published in partnership with the Sealy Institute for Vaccine Sciences npj Vaccines (2021) 89 N.G. Rouphael et al. 8 Study approval 25. Quan, F. S. et al. Long-term protective immunity from an influenza virus-like All participants provided written informed consent for participation in the particle vaccine administered with a microneedle patch. Clin. Vaccin. Immunol. study before enrollment. The study was approved by the Emory University 20, 1433–1439 (2013). and the Georgia Institute of Technology institutional review boards. 26. Quan, F. S., Kim, Y. C., Compans, R. W., Prausnitz, M. R. & Kang, S. M. Dose sparing enabled by skin immunization with influenza virus-like particle vaccine using microneedles. J. Control Release 147, 326–332 (2010). DATA AVAILABILITY 27. Rouphael, N. G. et al. The safety, immunogenicity, and acceptability of inactivated influenza vaccine delivered by microneedle patch (TIV-MNP 2015): a randomised, The data that support the findings from this study are available from the partly blinded, placebo-controlled, phase 1 trial. Lancet 390, 649–658 (2017). corresponding author on reasonable request. 28. Monto, A. S. et al. Antibody to influenza virus neuraminidase: an independent correlate of protection. J. Infect. Dis. 212, 1191–1199 (2015). Received: 23 February 2021; Accepted: 11 June 2021; 29. Couch, R. B. et al. Antibody correlates and predictors of immunity to naturally occurring influenza in humans and the importance of antibody to the neur- aminidase. J. Infect. Dis. 207, 974–981 (2013). 30. Monsalvo, A. C. et al. Severe pandemic 2009 H1N1 influenza disease due to pathogenic immune complexes. Nat. Med. 17, 195–199 (2011). 31. Bentebibel, S. E. et al. 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npj Vaccines (2021) 89 Published in partnership with the Sealy Institute for Vaccine Sciences N.G. Rouphael et al. 9 52. Vassilieva, E. V. et al. Improved immunogenicity of individual influenza vaccine A.S., Y.X., Y.Z., D.W., D.S., and S.K. optimized and performed immunologic assays. L.L., components delivered with a novel dissolving microneedle patch stable at room M.J.M., H.G., and F.K. supervised the immunologic assays. N.G.R. and M.R.P. supervised temperature. Drug Deliv. Transl. Res. 5, 360–371 (2015). the study. All authors approved the final version of the report. 53. Martínez-Sobrido, L. et al. Hemagglutinin-pseudotyped green fluorescent protein-expressing influenza viruses for the detection of influenza virus neu- tralizing antibodies. J. Virol. 84, 2157–2163 (2010). COMPETING INTERESTS fl 54. Rajendran, M. et al. Analysis of anti-in uenza virus neuraminidase antibodies in The authors declare the following competing interests: M.R.P. is an inventor on children, adults, and the elderly by ELISA and enzyme inhibition: evidence for licensed patents and has ownership interest in companies developing microneedle original antigenic sin. mBio 8, https://doi.org/10.1128/mBio.02281-16 (2017). products (Micron Biomedical). These potential conflicts of interest have been fl 55. Khurana, S. et al. Bacterial HA1 vaccine against pandemic H5N1 in uenza virus: disclosed and are overseen by Georgia Institute of Technology. S.H. and D.M. have an evidence of oligomerization, hemagglutination, and cross-protective immunity in ownership interest in a company developing microneedle products (Micron – ferrets. J. Virol. 85, 1246 1256 (2010). Biomedical). All other authors declare no competing interests. 56. Khurana, S. et al. Properly folded bacterially expressed H1N1 hemagglutinin globular head and ectodomain vaccines protect ferrets against H1N1 pandemic fl in uenza virus. PLoS ONE 5, e11548 (2010). ADDITIONAL INFORMATION 57. Khurana, S. et al. Recombinant HA1 produced in E. coli forms functional oligomers and generates strain-specific SRID potency antibodies for pandemic influenza Supplementary information The online version contains supplementary material vaccines. Vaccine 29, 5657–5665 (2011). available at https://doi.org/10.1038/s41541-021-00353-0. 58. Verma, S. et al. Oligomeric recombinant H5 HA1 vaccine produced in bacteria protects ferrets from homologous and heterologous wild-type H5N1 influenza Correspondence and requests for materials should be addressed to N.G.R. challenge and controls viral loads better than subunit by eliciting high-affinity antibodies. J. Virol. 86, 12283–12293 (2012). Reprints and permission information is available at http://www.nature.com/ 59. Crotty, S. et al. Cutting edge: long-term B cell memory in humans after smallpox reprints vaccination. J. Immunol. 171, 4969–4973 (2003). Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ACKNOWLEDGEMENTS Joanne Altieri-Rivera, Vinit Karmali, Regina Mosley, Haripriya Kalluri, Winston Pewin, Natalie J. Thornburg, Elena V. Vassilieva, Ioanna Skountzou, and Richard W. Compans. We also thank Maryna Eichelberger for making the H6NX viruses available to us. This Open Access This article is licensed under a Creative Commons study was funded by a grant from the National Institute of Biomedical Imaging and Attribution 4.0 International License, which permits use, sharing, Bioengineering (U01 EB012495); S.K. and A.S. received funding from a training grant adaptation, distribution and reproduction in any medium or format, as long as you give from the National Institute of Allergy and Infectious Diseases (T32 AI074492); and appropriate credit to the original author(s) and the source, provide a link to the Creative instrumentation support was provided to the Hope Clinic by the Georgia Research Commons license, and indicate if changes were made. The images or other third party Alliance. Work in the Krammer laboratory was partially funded by NIAID grant R01 material in this article are included in the article’s Creative Commons license, unless AI117287, NIAID CEIRS (contract HHSN272201400008C), and NIAID Collaborative indicated otherwise in a credit line to the material. If material is not included in the Influenza Vaccine Innovation Centers (CIVIC, contract 75N93019C00051). article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons. AUTHOR CONTRIBUTIONS org/licenses/by/4.0/. N.G.R., L.L., D.M.A., S.H., M.J.M., and M.R.P. contributed to the study concept and design. N.G.R. did the literature search. N.G.R. wrote the initial draft of the manuscript. N.G.R., L.L., S.T., M.P.M., T.W., and Y.K. analyzed and interpreted data. L.L., M.N., S.K., © The Author(s) 2021

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